Project Summary: Clathrin-mediated endocytosis (CME) is an essential pathway used by all eukaryotes for the transport of extracellular cargo into the cell. By controlling many of the signals that are transmitted between cells, CME is a key component in the development of organisms. Although the basic mechanism of clathrin-coated vesicle formation is known, an outstanding question remains, how is the transition from early clathrin coated structures to productive vesicles controlled? Productive vesicles are only produced from early structures about half of the time. Establishing the mechanisms whereby clathrin-coat remodeling can drive disassembly or vesicle formation is critical to understanding when cargo is internalized in healthy or diseased cells. The problem is a natural target for biophysical modeling because the fundamental structure of the problem (the clathrin cage) is known, but predicting how cargo uptake depends on the stoichiometry of the components, membrane bending, or ATP-expenditure is remarkably difficult because of the complexity of the process. We synthesize experimental data into a global model of CME that includes the full network of interacting components and tracks the spatial and temporal dynamics of each molecule as they diffuse, react, and assemble. In collaboration with expert cell biologists, our computational model will provide a quantitative and visual record of clathrin- coated vesicle formation. Our proposed work will determine physical requirements for disassembling clathrin-coated structures on membranes, and the coupling of membrane bending dynamics to clathrin-coated structure assembly with varying adaptor protein composition. Through construction of a comprehensive model of CME components, we test whether the activity of phosphatases in altering lipid composition at sites of clathrin-coated structures can trigger selective disassembly of sites lacking cargo. This proposed work will help determine the physical requirements for vesicle formation at fast (~ms) or slow (~seconds) time-scales, in distinct cell types. The impact of this proposal will be a validated, `whole-cell' type model of CME and powerful new software tools that will be publicly available for shared use. The software will be applicable to studying mechanisms of mutli-protein assembly and membrane remodeling not only in CME, but a wide range of cellular processes including cell division, cytoskeletal assembly, and viral budding.